The aim of this proposal is to elucidate the mechanism underlying the developmental regulation of the pyruvate carboxylase gene. A cloned cDNA for human pyruvate carboxylase will be used to isolate the expressed gene by screening a human genomic DNA cosmid library. The structure of the gene will be analyzed by conventional techniques such as restriction mapping, R-loop mapping and nucleotide sequencing. The cDNA will also be used to study the expression of the pyruvate carboxylase gene in differentiating mouse preadipocytes and in developing brain and liver tissues of the rat. The levels of pyruvate carboxylase mRNA during development will be quantitated by Northern blotting. The rates of transcription and turnover of the pyruvate carboxylase mRNA will be measured in the differentiating mouse cells. The 5' region of the gene will be thoroughly studied by identifying transcription initiation site(s), RNA splicing sites and promoter and enhancer-like elements. The goal of this research is to develop an understanding, at a molecular level, of the mechanisms involved in regulating the expression of a eukaryotic gene during development. Cellular RNA isolated from human mutant cell lines deficient in pyruvate carboxylase activity will be analyzed by S1 nuclease assays. The aim of this research is to identify unique genetic mutations that are involved in the processing of mitochondrial proteins. The pyruvate carboxylase gene from cell lines having mutations in the leader peptide region will be cloned and the mutations identified by DNA sequencing.